The Wnt signaling pathway plays a crucial role in embryonic development, in tissue regeneration and in many other cellular processes including cell fate, adhesion, polarity, migration, and proliferation. Dysregulated expression of components within the Wnt pathway triggers many diseases, and most importantly, heralds cancer (Klaus & Birchmeier, 2008).
Of the multiple known Wnt proteins, some preferentially trigger the well-characterized canonical pathway, which enhances the stability, nuclear localization and activity of β-catenin, and the downstream activation of genes targeted by the TCF/LEF transcription machinery. Other Wnts, e.g., Wnt5a deviate from this canonical paradigm, and trigger so-called non-canonical pathways (Kuhl et al, 2000; Niehrs, 2001; Winklbauer et al, 2001). Among other events, these non-canonical pathways induce the elevation of intracellular Ca2+ and activation of the small G proteins RhoA and Rac1, which regulate polarized cell movements and the planar polarity of epithelial cells (Kuhl et al, 2000; Mayor & Theveneau, 2014; Sheldahl et al, 1999). Of critical importance, non-canonical Wnt signaling antagonizes the canonical Wnt pathway (Ishitani et al, 2003; Olson & Gibo, 1998; Tones et al, 1996), although it is unclear how this occurs. Despite the lack of molecular mechanisms, dysregulation of the non-canonical Wnt pathway is widely believed to drive cancer via a two-faceted mechanism (McDonald & Silver, 2009)—1) non-canonical Wnt signaling suppresses tumorigenesis by antagonizing the canonical β-catenin/TCF/LEF pathway, and inhibition of non-canonical Wnt signaling heralds neoplastic transformation (Grumolato et al, 2010; Ishitani et al, 2003; Medrek et al, 2009); 2) hyperactivation of non-canonical Wnt signaling enhances cancer invasion/metastasis by activation of Rac1 and remodeling of the actin cytoskeleton (Yamamoto et al, 2009) and by upregulating CamKII and PKC (Dissanayake et al, 2007; Weeraratna et al, 2002). Little is known as to how such dysregulation of non-canonical Wnt signaling, i.e., early inhibition and late hyperactivation is orchestrated during cancer progression.
Non-canonical Wnt signaling is initiated by the binding of Wnt ligands to receptors of the Frizzled (FZDR) family. These receptors belong to the G protein-coupled receptor (GPCR) superfamily, which classically activate trimeric G proteins. However, the interplay between FZDR and G proteins in Wnt signaling is very controversial—on one hand, there is a wealth of evidence indicating that trimeric G proteins regulate Wnt signaling (Katanaev et al, 2005; Koval et al, 2011; Liu et al, 2005; Malbon, 2004; Schulte & Bryja, 2007). On the other hand, definitive evidence for the direct activation of trimeric G proteins by FZDR's is elusive. The experimental difficulties and controversies in the field have led to provocative speculations that FZDRs may not bind G proteins directly, but do so indirectly via other intermediates within the Wnt signaling pathway (Schulte & Bryja, 2007), but such intermediate ‘linker’ molecules have not been identified. Recent advances in the field of trimeric G protein signaling have important implications in this regard. It has become increasingly clear that the activity of trimeric G proteins is regulated by a plethora of accessory proteins (Bumer & Lanier, 2014; Sato et al, 2006; Siderovski & Willard, 2005) beyond classical activation by GPCRs. Among these accessory proteins, a subset of proteins called non-receptor Guanine nucleotide Exchange Factors (GEFs) are uniquely positioned to fulfill the role of an intermediate to trigger G protein signaling upon Wnt stimulation because they are cytoplasmic factors capable of activating G proteins (Garcia-Marcos et al, 2009; Garcia-Marcos et al, 2011b; Lanier, 2004; Lee & Dohlman, 2008; Natochin et al, 2005; Oner et al, 2013; Tall et al, 2003).
There is no single cytosolic target interface at the cross-roads of RTKs and Wnt receptors. There is no single target described in any G protein pathway that modulates β-Catenin signaling either. Further, there is no current tool that is useful in studying the endogenous Wnt receptor protein in cells/tissues.